The ability to produce recombinant human proteins has led to major advances in human health care and remains an active area of drug discovery. Many therapeutic proteins require the posttranslational addition of glycans to specific asparagine residues (N-glycosylation) of the protein to ensure proper structure-function activity and subsequent stability in human serum. For therapeutic use in humans, glycoproteins require human-like N-glycosylation. Mammalian cell lines (e.g., CHO cells, human retinal cells) that can mimic human-like glycoprotein processing have several drawbacks including low protein titers, long fermentation times, heterogeneous products, and continued viral containment. It is therefore desirable to use an expression system that not only produces high protein titers with short fermentation times, but can also produce human-like glycoproteins.
Fungal hosts such as the methylotrophic yeast Pichia pastoris has distinct advantages for therapeutic protein expression—e.g. it does not secrete high amounts of endogenous proteins, it has a strong inducible promoter, it can be grown in defined chemical media, and it can produce high titers of recombinant proteins (Cregg et al., 2000). However, glycosylated proteins expressed in P. pastoris contain additional mannose sugars resulting in “high mannose” glycans, as well as mannosylphosphate groups which impart a negative charge onto glycoproteins. Glycoproteins with either high mannose glycans or charged mannans are a high risk for illiciting an immune response in humans (Takeuchi, 1997; Rosenfeld and Ballou, 1974). Accordingly, it is desirable to produce therapeutic glycoproteins in fungal host systems, such that the pattern of glycosylation is identical or at least similar to that in humans.
Some fungal hosts contain immunogenic β-mannosylation on glycans of glycoproteins. In order to circumvent antigenicity, it is desirable to eliminate β-mannosylation in the production of human-like glycoproteins. Oligomannosides with β-1,2-linkage were first described by Shibata et al., (1985) in association with Candida albicans cell wall phosphopeptidomannan by phosphodiester bridges. Subsequently, three types of α-1,2 linkages have been identified in the side chains of Candida cell wall mannans. The first is a β-1,2-linked manno-oligomer located in a phosphodiesterified oligosaccharide moiety which is a common epitope in the mannans of several Candida species (Shibata et al 1993a). The second type is a β-1,2-linked mannose unit attached to the nonreducing terminal of the α-1,2 oligomannosyl side chains in the mannans of Candida albicans, tropicalis and glabrata (Kobayashi et al., 1989, 1992 and 1994). The third type of β-1,2 linkage is found in Candida guilliermondii and contains β-1,2 linked mannose units attached to an α-1,3 linked mannose unit (Shibata et al., 1993b).
Despite these findings, the studies on β-1,2 linkages have been limited by unsuccessful attempts to identify a β-1,2 mannosyl-transferase gene. Suzuki et al., (1997) characterized the presence of a β-1,2-mannosyltransferase in Candida guilliermondii, however, a gene for this enzyme has yet to be cloned.
In C. albicans yeast, both the β-oligomannosides which make up the acid-labile region of the phosphomannan complex, and α-oligomannosides, which make up the acid-stable region of the complex, serve as adhesins in the attachment of these pathogenic yeast cells to host splenic and lymph node macrophages (Cutler, 2001). Interestingly, antibodies protective against various forms of candidiasis recognize β-linked mannotriose, but not oligomannosides of greater mannose chain length (Han et al, 1997). It was reported that patients who develop deep tissue invasion with C. albicans, do not have detectable antibody titers specific for β-linked oligomannosides, whereas such antibodies were present in healthy individuals (Jouault et al, 1997).
There are few examples of β-linked mannose residues on glycoproteins from P. pastoris. In 1986, Kobayashi et al, described a modified acetolysis method with milder conditions for the isolation of manno-oligosaccharides composed predominantly of β-1,2 linked mannose residues. In 2003, Trimble et al reported the presence of β-1,2-linked mannose residues in the recombinant human bile salt-stimulated lipase (hBSSL) expressed in P. pastoris. As evidenced by the presence of protective antibodies in uninfected individuals, β-linked mannans are likely to be immunogenic. Additionally, exposed mannose groups on therapeutic proteins are rapidly cleared by mannose receptors on macrophage cells, resulting in low drug efficacy. Thus, the presence of β-linked mannose residues on N- or O-linked glycans of heterologous therapeutic proteins expressed in a fungal host e.g., P. pastoris is not desirable given their immunogenic potential and their ability to bind to clearance factors.
What is needed, therefore, is a method for removing undesired mannose residues on glycoproteins for the production of therapeutic glycoproteins.